Over expression of single-stranded molecules

ABSTRACT

A method of synthesis of new and useful single-stranded DNAs which have a stem-loop configuration (ss-slDNA). The method is an in vivo or an in vitro synthesis. Replicating vehicles which produce these ss-slDNAs. The ss-slDNAs are described. Uses for these slDNAs are disclosed. They can be used for introducing random mutations, they lend themselves for replication by a variant of the PCR method. They can also be used for regulating gene function. Other uses are disclosed.

This is a 371 application of PCT/US94/02169, filed on Mar. 1, 1994,which is a continuation-in-part of pending application Ser. No.08/284,860, filed on Aug. 2, 1994, which is a Continuation in Part of08/024,676, filed on Mar. 1, 1993, which is a Continuation in Part of07/753,111, filed on Aug. 30, 1991, abandoned. Both of these patentapplications (parent applications) are explicitly incorporated hereinword for word as if each had been fully reproduced hereinafter.

FIELD OF THE INVENTION

The invention concerns the field of recombinant DNA. More particularly,the invention relates to a method of synthesis of new and usefulsingle-stranded DNA which have a stem-loop configuration (ss-slDNA). Theinvention relates to an in vitro and in vivo method of synthesis.Further, the invention relates to the replicating vehicle which producethese ss-slDNAs. Moreover, the invention relates these novel structuresand discloses uses for these structures. There is described a method foramplifying ss-slDNAs with or without genes encoding a target protein.Moreover, the invention discloses a method for regulating gene functionby the use of ss-slDNA.

BACKGROUND

Duplication of part of the genome is known to occur via an RNAintermediate which is reverse-transcribed into complementary DNA (cDNA)by reverse transcriptase. For a review, see Weiner et al., Ann. Rev.Biochem., 55, 631 (1986). The consequential reverse flow of geneticinformation is considered to have played a major role in theevolutionary diversification of eukaryotic genomes. A similar mechanismmay very well have been responsible for genomic evolution in procaryotesin the light of the recent discoveries of bacterial transcriptases. SeeInouye and Inouye, TIBS, 16, 18 (1991a) and Inouye and Inouye, Ann. Rev.Microbiol., 45, 163 (1991b). Gene duplication through cDNA synthesis byreverse transcriptase is believed to have played an important role indiversification of genomes during evolution.

The invention arose in connection with basic research related to genomeevolution. The synthesis of a unique ss-slDNA during plasmid DNAreplication was demonstrated. It may be speculated that slDNA productionmay be widely prevalent during both procaryotic as well as eucaryoticchromosomal DNA replication. The chromosomal genetic elements followedby IR structures may always be subject to duplication into slDNA at afrequency depending on the stability of the IR structure and theproperty of the polymerase(s).

It has been shown that there are many inverted repeat (IR) structures(approximately 1,000 copies in E. coli), known as REPs for repetitiveextragenic palindromic sequences or PUs for palindromic units. Higginset al., Nature, 298, 760 (1982), Gilson et al., EMBO. J., 3, 1417 (1984)and Gilson et al., Nucl. Acids Res., 19, 1375 (1991). These structuresappear to be associated with specific cellular components including DNApolymerase I, and may be playing a significant role in chromosomalorganization. Gilson et al., Nucl. Acids Res., 18, 3941 (1990) andGilson et al., EMBO. J., 3, 1417 (1984). It should also be noted thatapproximately 6% of the human genome are occupied with elements calledAlu whose transcriptional products have been shown to containsubstantial secondary structures. Simmett et al., J. Biol. Chem., 266,8675 (1991).

Since slDNA synthesis does not require RNA intermediates nor reversetranscriptase activity in contrast to that of cDNA synthesis, slDNA maybe more frequently produced than cDNA. Thus, slDNAs might have played amajor role similar to cDNA in the genomic evolution of both procaryotesand eucaryotes by duplicating genetic elements which then were dispersedor rearranged within the genome.

SUMMARY OF THE INVENTION

The parent applications relate to processes for synthesizing a novelsingle-stranded DNA structure, slDNA, in vivo or in vitro, to the slDNAstructures and to various other genetic constructs. The parentapplications also relate to slDNAs which carry an antigene in thesingle-stranded portion of the slDNA, which can be an antisense fragmentwhich binds to a target mRNA and inhibits mRNA translation to protein orbinds to double-stranded (ds)DNA, thereby forming a triple helix whichinhibits the expression of target DNA.

The present invention relates to the over-expression of single-strandedDNA molecules, more particularly slDNAs in yields heretofore neveraccomplished, to the method of such production and to the geneticconstructs to achieve these objectives.

The slDNA structures (as described hereinabove) are single-stranded DNAmolecules comprising a single-stranded loop and a tail of duplexedsingle-stranded complementary bases ending with one single 5' and onesingle 3' termini, which may have a single-stranded overhang withrespect to each other.

In view of the fact that single-stranded molecules are notoriouslyunstable, it was unexpected that such single-stranded structures couldbe produced in such high yields in accordance with the invention.

In accordance with the present invention, a fundamental finding has beenmade. It has been found that portions of the genome can be directlyduplicated from the genome. This gene duplication it has been found,requires neither an RNA intermediate nor reverse transcriptase andoccurs during DNA replication.

Briefly described, the invention provides a method (or process) forsynthesizing a novel and useful single-stranded DNA (ssDNA) molecule.The method involves using a DNA inverted repeat (IR) and necessarycomponents to start and synthesize a single-stranded structure. Theinvention also provides a system for synthesizing such ssDNA from andwith the necessary components including a DNA inverted repeat. Theinvention further provides competent replicating vehicles which includeall the necessary elements to synthesize the novel ssDNA molecules. Theinvention also provides novel and useful ssDNA molecules which form aunique structure. The structure has a stem constituted of duplexed DNAof complementary bases; the stem terminates at one of its ends by twotermini, respectively a 3' and 5' terminus, and at the other end by thessDNA forming a loop.

The invention contemplates carrying out the method and the systems invitro or in vivo.

Various uses of the new molecules are described, including a method forproviding random mutations in genes with the aim of generating proteinswith improved or novel biological properties. Another interestingcontemplated use is to integrate the ssDNA molecules of the inventioninto DNA to generate a triplex DNA of increased stability. Another usefor the ssDNA of the present invention is as antisense DNA. Another useis the application of the polymerase chain reaction (PCR) using a singleprimer.

The invention and its several embodiments are described in greaterdetail hereinafter.

By the method of the invention there is produced a ssDNA molecule whosestructure comprises a stem portion of duplexed DNA of annealedcomplementary bases within the ssDNA, which stem forms, at one of itsends, the 5' and 3' termini of the ssDNA molecule and, at the other end,a loop, which is constituted of a single-strand of non-annealed baseswhich joins the two single-strands of the stem. In a specificembodiment, strand ending in the 3' terminus is longer than the otherstrand. The slDNAs may include DNA segments capable of encoding aprotein, more particularly any gene(s). The gene will be located betweenthe loop and the termini. The gene may be a gene carrying a mutation(s).

A mechanism postulated for the synthesis of slDNAs is illustrated inFIG. 6A and B. The synthesis is believed to involve, in summary, thefollowing course: DNA is initiated at the origin of replication (OR),replication of a first strand (or "the" or "a" strand) then proceedsusing one of the strands of the double-stranded DNA as template (by thesame mechanism as chromosomal DNA replication (see Tomizawa et al.,Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), proceeding through the IRstructure resulting in the replication of the entire plasmid genome whena plasmid is used. However (as is described in greater detailhereinafter), part of the first strand forms a loop structure as thefirst strand synthesis is disrupted or terminated within the IR (FIG. 6,from steps 2 to 3). The loop forms a short duplexed region whichfunctions as priming site for continuing DNA synthesis. DNA chainelongation resumes from the newly formed 3' end, now forming the secondstrand (or "the other" strand) utilizing the nascent first strand as atemplate. An alternative (or concurrent) postulated synthesis course isdescribed hereinafter. Thus, the direction of DNA synthesis is reversedby template switching to duplicate the DNA fragment (FIG. 6, from steps4 to 5). The newly synthesized slDNA molecule dissociates itself fromthe parent template strands which undergo another round of replicationto form another slDNA. The slDNA is isolated, if necessary, purified.

In another embodiment of the invention, there is provided a DNAself-replicating vehicle, e.g., a plasmid into which there has beeninserted a DNA fragment which contains an inverted repeat (IR)structure, the DNA fragment which will serve as template for synthesisof the ssDNA and a suitable priming site, e.g., an origin of replication(OR), such as the E. coli origin of replication (ColE1). The IR issituated downstream of the OR. The self-replicating vehicle isreplicated in a suitable host, e.g., an E. coli. The host will containat least one DNA polymerase to synthesize the ssDNA from the template.In this specific embodiment, it is presumed that there are twopolymerases, a first DNA polymerase contributing to the elongation of aportion of the ssDNA from the OR to the IR, the first strand, and asecond DNA polymerase which synthesizes the balance or second strand ofthe ssDNA strand. As the synthesis of the first strand terminates,complementary bases anneal to form a single-stranded non-annealed loop.The synthesis of the second strand then takes place. A new DNA structureis synthesized which is constituted of a duplexed DNA stem and asingle-stranded loop structure at the opposite end. For this new DNAstructure the term "stem-loop" or "slDNA" has been coined.

Another specific embodiment provides a replicating vehicle in which apromoter, specifically the lac promoter-operator has been deleted. AnslDNA was nonetheless produced, supporting the synthesis model proposed,as described further herein.

The plasmid may be constructed to contain any selected DNA sequencecapable of encoding a protein between the priming site and the IR inwhich event the slDNA synthesized will contain the DNA sequence or amutation thereof.

This invention provides a method for regulating gene function by the useof slDNA.

The slDNAs of the invention need not be synthesized by aself-replication vehicle, but may be synthesized in an appropriate invitro system constituted of a any segment of DNA, linear or not, whichcontains any IR and the necessary elements to synthesize the ssDNA. Suchsystem is described hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a plasmid of the invention, pUCK106.

FIG. 2 shows the DNA sequence of 215-bp inserted at the XbaI site ofpUCK19.

FIGS. 3A-3C shows an ethidium bromide staining of polyacrylamide gel ofthe production of an slDNA from pUCK106 and its characteristics.

FIGS. 4A-4C shows an autoradiograph of a dried polyacrylamide gel ofdimer formation of the slDNA from pUCK106.

FIGS. 5A-5E illustrates determination of the DNA sequence of the slDNAfrom pUCK106.

(A) shows an autoradiograph of a dried polyacrylamide gel of thedetermination of the DNA sequence of the 5' end region of the slDNA.

(B) shows an autoradiograph of a dried polyacrylamide gel of the 5' endsequencing of the loop region of the slDNA.

(C) shows an autoradiograph of a dried polyacrylamide gel of the 3' endsequencing of the loop region of the slDNA.

(D) shows an autoradiograph of a dried polyacrylamide gel of the DNAsequence of the 3' end region of the slDNA.

(E) shows the structure of the slDNA from pUCK106.

FIGS. 6A-6B illustrates two possible models of slDNA synthesis.

FIG. 7, lanes 1 and 3 show the HaeIII digest of pBR322 as size markers.Lane 2 shows the slDNA from the preparation from pUC7.

FIG. 8 shows the gene for β-galactosidase in a transformed vector.

FIG. 9 shows plasmids pUCK106d.P.O., pUCK106d.P.O.-I, andpUCK106d.P.O.-II.

FIG. 10 shows the construction of plasmid pXX566.

FIG. 11 shows the relation between ampicillin concentration and colonynumber in example 9-3.

FIG. 12 shows plasmids pUCKA106d.P.O., pUCKAGT37, and pUCKACA37.

FIG. 13 shows the DNA sequence inserted into pMS434 and the location ofthe primers.

FIG. 14 shows the construction of plasmids pYES2-8-106, and pYES2-9-106.

FIG. 15 shows the DNA fragments produced by digestion of pYES2-8-106,pYES2-9-106, and slDNA with SalI.

FIG. 16 shows the location of three primers for detecting slDNA.

FIG. 17 illustrates the schematic for constructing a replicable vehicleof the invention for over-expressing slDNAs.

FIG. 18 illustrate a final construct, pHS2870.

FIG. 19 illustrates the schematic for constructing construct pHS2870(˜480 bp), showing the insertion at restriction site HindIII, offragment HindIII of 49 bases and plasmid named pHS2870GT (˜500 bp).

FIG. 20 illustrates gel electrophoresis of slDNAs extracted from pHS2870and from pHS2870GT.

FIGS. 21A-21B illustrates the construction of E. coli MC4100 λpFTR1F'.

FIG. 22 shows the detection of expression level of β-galactosidase.

DEPOSIT OF GENETIC MATERIAL

Plasmid pUCK106 has been deposited in accordance with the BudapestTreaty with the American Type Culture Collection (ATCC) under AccessionNo. 68679.

Plasmid pUCK106Δlac^(PO) has been deposited in accordance with theBudapest Treaty with the ATCC under Accession No. 68680.

EXAMPLES

The following examples are offered by way of illustration and are notintended to limit the invention in any manner. In these examples, allpercentages are by weight if for solids and by volume for liquids, andall temperatures are in degrees Celsius unless otherwise noted.

For convenience and clarity, the Examples refer to and provide also adetailed description of the Figures.

Example 1

FIG. 1 illustrates pUCK19 (circular map), a specific plasmid made asshown below. The open bar in the circular map represents thekanamycin-resistant gene (from Tn 5). The straight open bar shown at theupper right hand side represents a 215-bp DNA fragment inserted at theXbaI site, which contains 35-bp inverted repeat (IR) sequences. Solidarrows show the IR structure. The solid circle design is the origin ofreplication (Ori). The longer open arrow indicates the direction of DNAreplication from the OR. The smaller open arrow shows the position ofthe lac prompter-operator (lac^(PO)).

FIG. 2 shows the DNA sequence of 215-bp inserted at the XbaI site ofpUCK19 consisting of DNAs shown as SEQ ID Nos. 1 and 2 in the sequencelisting, respectively. This plasmid is designated herein as pUCK106. Theopen arrows indicate the IR sequences. The HindIII (AAGCTT) site showsthe center of the IR.

Mismatched positions in the IR are shown by two open spaces in thearrows with mismatched bases C and T inserted in the spaces.

The DNA fragment discussed above (which contains the IR) has thefollowing sequence shown as FIG. 2.

Example 2

This example illustrates the production of an slDNA from pUCK106 and itscharacteristics.

(A) E. coli CL83 was transformed with either pUCK19, pUCK106 and withpUCK106Δlac^(PO) and a plasmid DNA fraction was prepared. A DNApreparation (after ribonuclease A treatment) was applied to a 5%acrylamide gel for electrophoresis. The gel was stained with ethidiumbromide. With reference to FIG. 3A, lane 1 shows the HaeIII digest ofpBR322 as size markers. The numbers shown are base pairs; lane 2, theDNA preparation from cells harboring pUCK19; lane 3, pUCK106; and lane4, pUCK106Δlac^(PO).

pUCK19 is a kanamycin variant of pUC19.

(B) The slDNA from pUCK106 was purified by polyacrylamide gelelectrophoresis followed by various restriction enzyme digestion. Thedigests were analyzed by polyacrylamide (5%) gel electrophoresis, andthe gel was stained by ethidium bromide. With reference to FIG. 3B, lane1, the HaeIII digest of pBR322 as size markers; lane 2, slDNA withoutdigestion; lane 3, slDNA digested with XbaI; lane 4, with HindIII; andlane 5, with PvuII.

(C) Heat-denaturation of the slDNA from pUCK106. The purified slDNA (asdescribed above) was solubilized in 10 mM Tris-HCl (pH 8.0) and 1 mMEDTA. The slDNA solution was incubated in a boiling water bath for 3minutes and quickly chilled in an ice bath. Samples were analyzed asdescribed in A. With reference to FIG. 3C, lane 1, the HaeIII digest ofpBR322 as size markers; lane 2, slDNA without heat treatment; and lane3, slDNA heat-denatured followed by quick cooling.

The slDNA has a loop of 53 bases, a stem of about 440 bases and anoverhang 3' tail of 15-18 bases.

Example 3

This example illustrates dimer formation of the slDNA from pUCK106.

(A) The purified slDNA from pUCK106 as described in FIG. 2 wassolubilized in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM MgCl₂. TheslDNA solution was incubated in a boiling water bath for 3 minutes andthen gradually cooled. The renatured slDNA was digested with XbaI andthe DNA fragments thus generated were labeled at their 5' ends with γ-³²P!ATP and T4 polynucleotide kinase. These products were applied to a 5%polyacrylamide gel. After electrophoresis, the gel was dried andsubjected to autoradiography.

With reference to FIG. 4A, lane 1, the HaeIII digest of pBR322 as sizemarkers; lane 2, the EcoRI and HindIII digest of λ DNA as size markers.Numbers shown are in base pairs; lane 3, the slDNA from pUCK106 withouttreatment; lane 4, the XbaI digest of the untreated slDNA; lane 5, theslDNA after heat-denaturation followed by gradual cooling; and lane 6,the XbaI digest of the slDNA from lane 5. Bands are marked from "a" to"e" at the right hand side.

(B) Characterization of fragment "d" in FIG. 4A. Fragment "d" purifiedfrom the gel; lane 3, the HindIII digest of the purified fragment "d";and lane 4, the purified fragment "d" was heat-denatured and quicklychilled as described with respect to FIG. 3.

(C) Schematic representation of bands "a" to "e" shown in A and B (FIG.4C). X and H represent XbaI and HindIII sites, respectively. These aretwo other HindIII sites in fragment "a" very close to the XbaI sites(within fragment "c"). These HindIII sites are not shown.

Example 4

This example illustrates determination of the DNA sequence of the slDNAfrom pUCK106.

(A) Determination of the DNA sequence of the 5' end region of the slDNA.0.2 μg of the isolated and purified slDNA was used for sequencing by thechain termination method. Primer "a" (^(5') GGTTATCCACAGAATCAG^(3')),shown as SEQ ID No. 3 in the sequence listing which corresponds to thesequence 96-bp downstream from the origin (see FIG. 5B) was used asprimer.

(B) 5' end sequencing of the loop region of the slDNA. 0.5 μg of theslDNA was digested with SacII and the DNA fragments thus generated werelabeled at the 5' end with γ-³² P!ATP and T4 polynucleotide kinase. TheDNA fragment migrated at approximately 40-bp was isolated and sequencedby the Maxam-Gilbert method.

(C) 3' end sequencing of the loop region of the slDNA. The SacII digestslDNA was labeled at the 3' end with γ-³² P!dideoxyATP using terminaldeoxynucleotidyl transferase. The DNA fragment containing the loopregion was isolated and sequenced by the Maxam-Gilbert method.

(D) DNA sequence of the 3' end region of the slDNA. The slDNA wasdigested with AflIII (see FIG. 5E). The 5' ends were labeled with γ-³²P!ATP and T4 polynudeotide kinase. The labeled products were separatedby a sequencing gel. The single-stranded DNA which migrated at 76 baseswas isolated and sequenced by Maxam-Gilbert method. By reference to FIG.5D the numbers represent the residue numbers from the origin of pUCK19.

(E) Structure of the slDNA from pUCK106. The slDNA consists of asingle-stranded DNA of 1137 to 1139 bases. The 5' end of the slDNAappears to be heterogeneous; some start from +1 while other start from-1, +2 and +3. The +1 position corresponds to the origin of ColE1 DNAreplication. Thus, various slDNAs have different length 5' endingstrands. At the 3' end a sequence of 16 bases is extended beyond the +1position of the 5' end. The loop is considered to be formed with the 4base sequence (AGCT) corresponding to the sequence at the center of theIR structure, where a HindIII site (AAGCTT) is designed to be placed.The slDNA has a tail of about 400 bases. The base pair corresponding tothe mismatch in the IR structure in pUCK106 was converted from C•T (inpUCK106) to C•G (in the slDNA) is shown between the SacII and PstIsites. The position of primer "a" used for DNA sequencing in FIG. 5A isshown by an arrow.

slDNAs can be constructed with stems of 300-4,000 or more bases. Theoverhang can be adjusted to any length desired, like from about 10 to80, as for example to about 50. The length should preferably not be tothe sacrifice of stability. The slDNAs will be constructed with loops ofthe desired sizes.

Separation and purification of the slDNAs are performed according tostandard techniques as by following the procedures described inMolecular Cloning, A Laboratory Manual, Sambrook et al., 2d Ed.(Sections 1.121-1.40) ("Sambrook").

Example 5

This example illustrates two possible models of slDNA synthesis (seeFIG. 6). The double-stranded DNA around the origin of the ColE1 DNAreplication is shown on the top. The shaded circle represents the DNAreplication complex which initiates DNA replication from the origin. Theopen arrows on the DNA strand indicate the position of the 35-bpinverted repeat (IR) structure (see FIG. 2) in the DNA sequence. Themismatched base pair (C•T) in the IR structure is also indicated withinthe arrows.

At step 1, the DNA replication fork proceeds from the origin (+1position) to the position indicated by the shaded circle. The newlysynthesized first strand is shown extending from the origin (a solidcircle) to the replication fork. The DNA replication complex reachesimmediately before the mismatched T residue in the IR structure that isshown by solid arrows. At step 2, the 3' end of the nascent stranddetaches from the DNA replication complex and a secondary structure isformed by the IR structure. At step 3, DNA synthesis reinitiates fromthe 3' end of the stem-loop structure utilizing either the nascentstrand (model A) or the upper parental strand (model B) as template. Atstep 4, DNA synthesis proceeds beyond the origin by 16 bases.

In model A, the primer RNA which remains attached at the 5' end of theDNA may be used as the primer RNA. Subsequently, the RNA may be removedresulting in the formation of slDNA. In model B, DNA synthesisterminates at the terH site by a similar mechanism known for thetermination of the second strand DNA synthesis.

It is conceivable that both models A and B can explain the synthesis,and the synthesis may proceed by both routes concurrently, at least forpart of the time. Thus, an appropriate template will be used for thesecond strand other than the strand which was the template for the firststrand.

Example 6 Construction of pUCK106Δlac^(PO)

When the 199-bp PvuII-HincII fragment containing the lacpromoter-operator was deleted from pUCK106 (see FIG. 1), the resultingpUCK106Δlac^(PO) produced a new slDNA which migrated faster than theslDNA from pUCK106 as shown at position (b) in lane 4, FIG. 3A. The sizeof this new slDNA was 360-bp in length, which is shorter than thepUCK106 slDNA by a length nearly identical to the size of the deletionin pUCK106Δlac^(PO).

In FIG. 3A, lane 3 shows the slDNA from the DNA preparation from cellsharboring pUCK106Δlac^(PO).

This experiment supports the model for slDNA synthesis proposed aboveand also indicates that the lac promoter-operator is not essential forslDNA synthesis. This notion was further supported by the fact that theaddition of isopropyl-β-D-thiogalactopyranoside, an inducer of lac, didnot affect the production of the slDNA from pUCK106. However, the reasonfor the reduction of slDNA synthesis from pUCK106Δlac^(PO) is not knownat present.

Example 7

The synthesis of slDNA was not dependent upon the primary sequence ofthe IR structure used for pUCK106. Interestingly, the pUC7 vector byitself, which has an IR structure at the polylinker site is also able toproduce an slDNA corresponding to the DNA fragment from the origin tothe center of the polylinker site.

The isolated and purified slDNA produced from pUC7 was 252-bp in length.A plasmid fraction was prepared and treated as in Example 2 and applied(and stained) to acrylamide gel for electrophoresis.

In FIG. 7, lanes 1 and 3 show the HaeIII digest of pBR322 as sizemarkers. Lane 2 shows the slDNA from the preparation from pUC7.

The slDNA of pUC7 was amplified by PCR (which is described in furtherdetail herein) and lane 4 (at arrow) shows the slDNA.

Example 8 Confirmation of the slDNA Structure

The slDNA structure and mechanism described above (illustrated in FIG.6) was confirmed as follows.

The DNA fragment that was synthetically constructed was providedintentionally with mismatched bases CT for CG. See FIG. 2 in the openspaces of the IR. After synthesis of the slDNA (with the second strandsnapped-back over the first strand) me mismatch has been repaired. FIG.5E, now CG appears. If the structure were not a snap-back structure, thepolymerase would have read right through the IR, would have read the Tand inserted an A; the new strand would still have contained themismatch. To replace the mismatched T, that IR portion necessarily hadto snap-back onto the first strand, thus allowing the polymerase to usethe first synthesized strand as template to synthesize the second strandand as it synthesizes it, insert the complementary base G in place ofthe mismatched T. This unequivocally establishes the synthesis mechanismand structure of the slDNAs of the invention.

Example 9

This example illustrates regulating gene function by the use of slDNAcontaining an antisense sequence (antisense slDNA).

9-1 Construction of Plasmids Which Produce slDNA

pUCK106d.P.O. was prepared from pUCK106 by the deletion of the 199-bpPvuII-HincII fragment containing the lac promoter-operator (FIG. 9).Then, the oligonucleotides with the structures shown as SEQ ID No. 4(antisense) and No. 5 (sense) in the sequence listing were synthesized,annealed, and inserted at the HindIII site of the pUCK106d.P.O. Theplasmid which produced the slDNA containing sense sequence (sense slDNA)was named pUCK106d.P.O.-I and the plasmid which produced antisense slDNAwas named pUCK106.d.P.O.-II (FIG. 9).

The slDNA is determined to have a loop of 42 bases, a stem of 360duplexed bases and a tail or overhang of 15-18 bases.

9-2 Preparation of the Host E. coli Which Contains the β-lactamase Gene

The EcoRI-HaeII fragment of approximately 1.6-kb containing theβ-lactamase gene from pBR322 (Takara Shuzo) was blunt-ended and insertedat the SmaI site of pHSG399 (Takara Shuzo). The resulting plasmid wasnamed pXX555. The 1695-bp EcoRI-HindIII fragment from pXX555 wasinserted at the EcoRI-HindIII site of the pXX325 derived from miniFplasmid see Proc. Natl. Acad. Sci. USA, 80, 4784-4788, 1983!. Theresulting plasmid was named pXX566 (FIG. 10). The E. coli MC4100,containing the β-lactamase gene, was prepared by transformation withpXX566. The transformant was named MC4100/pXX566.

9-3 Reduction of β-lactamase Gene Expression by Antisense slDNA

E. coli MC4100/pXX566/I and E. coli MC4100/pXX566/II were prepared fromE. coli MC4100/pXX566 by transformation with pUCK106d.P.O.-I andpUCK106d.P.O.-II, respectively. Each transformant was inoculated intoL-broth containing 50 μg/ml kanamycin and incubated overnight at 37° C.The culture was diluted 1:10000 with L-broth and 50 μl of each dilutionwas plated out on agar L-broth plates containing 50 μg/ml kanamycin and50-150 μg/ml ampicillin. After overnight incubation at 37° C., thenumber of colonies was counted. Table 1 and FIG. 11 show the relativepercent of the numbers (X-axis: ampicillin concentration, Y-axis: therelative percent, with 100% at the concentration of 50 μg/mlampicillin). The increase of relative death rate showed that β-lactamasegene expression was reduced by antisense slDNA.

                  TABLE 1                                                         ______________________________________                                        Ampicillin conc.                                                                           MC4100/pXX566/I                                                                            MC4100/pXX566/II                                    (μg/ml)   (sense)      (antisense)                                         ______________________________________                                        50           100          100                                                 75           83           60                                                  100          55           22                                                  150          0            0                                                   ______________________________________                                    

9-4 Measurement of β-lactamase Expression by Western Blotting

Each single colony of MC4100/pXX566/I and II as in Example 9-3, wasselected at random and inoculated into 5 ml of L-broth containing 20μg/ml ampicillin and 50 μg/ml kanamycin. After overnight incubation at37° C., 100 μl of each culture was inoculated into the same fresh media.Cells were harvested at the time when the absorbance at 600 nm reached1.2, washed with 10 mM sodium phosphate buffer (pH 7.1), and resuspendedin 1 ml of the same buffer. The cell density was recorded by measuringthe absorbance at 600 nm. The sonicated lysates of 5.36×10⁸ cells wereelectrophoresed on a 15% SDS-polyacrylamide gel, and the separatedproteins were transferred to a piece of PVDF membrane filter (Immobilon,Millipore). The filter was subsequently exposed to rabbitanti-β-lactamase polyclonal antibody (5 Prime-3 Prime, Inc.) anddetection was done With Immuno-Stain-Kit (Konica). After treatment withchromogenic substrate, colored bands appeared at their proper positions.Those bands indicated that the level of β-lactamase activity in thecells producing antisense slDNA was 30% lower than that in the cellsproducing sense slDNA.

Further, it was demonstrated that the gene copy of the sense and theantisense slDNAs are the same and that neither molecule containsdeletions or rearrangements. Photographs of gels containing the senseand the antisense slDNAs show that the slDNAs produced are the expectedsize suggesting that no gene rearrangement occurred in these constructs.In addition, the intensity of the sense and antisense slDNA bands issimilar, which shows that the copy number of the two are also similar.

These results are further supported by the results of the studies on thekanamycin resistant gene. The kanamycin gene expression is independentof the antisense system and hence serves as an internal control.Polyacrylamide gels of extracts derived from cells expressing the senseand antisense slDNAs show that the intensity of the bands representingthe Km resistance protein is the same. In addition, the total amount ofprotein in these two extracts except for β-lactamase is the same. Theseresults show that it is the antisense effect which is being demonstratedin the data presented. Thus, the changes in β-lactamase expressionobserved do not occur as a result of a global effect on celltranscription and translation, but is due directly to the expression ofthe antisense slDNA.

Example 10

This example illustrates slDNA capable of forming triple helix (triplehelix-forming slDNA).

10-1 Construction of pUCK106Ad.P.O.

The 106-NdeI sequence, which consists of the sequences shown as SEQ IDNos. 6 and 7 in the sequence listing, was inserted at the NdeI site ofthe pUCK19. The resulting plasmid was named pUCK106A. Plasmid pUCK106Ad.P.O. was constructed by the deletion of 206-bp HincII-VspI fragmentcontaining the lac promoter-operator region from pUCK106A.

10-2 Preparation of the Triple Helix-forming slDNA

The oligonucleotide shown as SEQ ID No. 8 (GT37 sequence) in thesequence listing and the oligonucleotide shown as SEQ ID No. 9 (CA37sequence) in the sequence listing were synthesized and phosphorylated at5' end. The duplexed oligonucleotide was made by annealing with theseoligonucleotides and inserted at the HindIII site of pUCK106Ad.P.O. inthe middle of 106-NdeI sequence. Each plasmid that produced the slDNAcontaining GT37 sequence or CA37 sequence was named pUCKAGT37 orpUCAKCA37, respectively (FIG. 12). E. coli MC4100 were transformed bythese plasmids, and each transformant was named MC4100/pUCKAGT37 orMC4100/pUCKACA37. These transformants and MC4100/pUCK106Ad.P.O.constructed in Example 9-2 were cultured in L-broth containing 70 μg/mlkanamycin. DNA was prepared by the alkaline-SDS method and treated withRNaseA. The slDNA was purified by polyacrylamide gel electrophoresis.

10-3 Preparation of the Target DNA

The oligonucleotide shown as SEQ ID No. 10 in the sequence listing andthe oligonucleotide shown as SEQ ID No. 11 in the sequence listing weresynthesized and phosphorylated at 5' end. The duplexed oligonucleotidewas made by annealing these oligonucleotides and inserting at theXhoI-HindIII site of pMS434 see Gene, 57, 89-99, (1987)!. This plasmidwas named pMSTR37.

The target DNA of triple helix formation was amplified by PCR withprimer M4 shown as SEQ ID No. 12 in the sequence listing and primer MS1shown as SEQ ID No. 13 in the sequence listing as primers and pMSTR37 asa template.

10-4 Triple Helix Formation on slDNA

The slDNA prepared in Example 10-2 was labelled with γ-³² P!ATP byphosphorylation. Triple helix was formed by mixing radiolabeled slDNAand excess target DNA in a 0.15M NaCl/10 mM MgCl₂ /5 mM tris-acetate(pH7.0) buffer, and then incubating overnight at 37° C. Triple helixformation was detected by 12% polyacrylamide gel electrophoresis in 50mM tris-borate/5 mM MgCl₂ (pH8.3) buffer.

As a result, triple helix formation was detected only with the slDNAcontaining GT37 sequence. On the other hand, triple helix formation wasnot detected with either slDNA containing CA37 sequence or intact slDNA.

Example 11

This example illustrates the synthesis of slDNA in Saccharomycescerevisiae.

11-1 Construction of Plasmids pYES2-8-106 and pYES2-9-106

The shuttle vector pYES2 (Invitrogen) containing ColE1 and 2μ originswas modified as illustrated in FIG. 14 and the derivatives were used toinvestigate whether slDNA can be produced in yeasts. First, the multiplecloning sites and the Gal I promoter region were removed from pYES2 bydigestion with MluI and SspI. Next, the remaining DNA fragment wasblunt-ended by T4 DNA polymerase and then self-ligated (step 1). Second,the DNA fragment A or B, shown as SEQ ID No. 14 or 15 in the sequencelisting, respectively, was inserted at the blunt-ended AvaI site of theplasmid of step 1 (step 2). Finally, the 227-bp BamHI-SalI DNA fragmentfrom pUCK106 was inserted at BamHI-SalI site of the plasmids of step 2(step 3). The resulting pYES2 derivatives were named pYES2-8-106 (leftside of FIG. 14) and pYES2-9-106 (right side in FIG. 14), respectively.

11-2 Purification of Plasmid DNA and slDNA From the Transformants

Saccharomyces cerevisiae YPH499 cells (Stratagene) were transformed bypYES2-8-106 or pYES2-9-106 by the LiAc procedure see Journal ofBacteriology, 153, 163-168, (1983)!. These transformants were grown on100 ml of YPD medium (1% yeast extract, 2% bacto-peptone, 2% dextrose)to an OD₆₀₀ of approximately 1.5. The cells were harvested bycentrifugation, and washed once with 10 ml of SCE solution (182 g/lSorbitol, 29.4 g/l Na₂ Citrate, 22.3 g/l Na₂ EDTA). Next, the cells weresuspended in solution I (SCE solution containing 0.1% β-mercaptoethanoland 0.5 mg/ml Zymolyase 100T) and shaken gently at 37° C. for 2 hours togenerate spheroplasts. Next, 8 ml of solution II (0.2N NaOH, 1% SDS)were added and the suspensions were held on ice for 5 minutes. Next, 6ml of solution III (60 ml of 5M K-acetate, 11.5 ml of glacial aceticacid, 28.5 ml of H₂ O) were added, held on ice for an additional 5minutes, and then centrifuged. The DNAs were precipitated by addition ofisopropanol to the supernatants, washed with 70% ethanol, andresuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.6). The DNAswere further purified by phenol/chloroform extraction, ethanolprecipitation, and using Qiagen tip 5 (Qiagen Inc.). Finally, thepurified DNAs were suspended in 20 μl of TE buffer.

11-3 Separation of the slDNA From the Plasmid DNA

10 μl of the samples obtained in Example 11-2 were treated with SalI. Bythis process, the plasmid DNA should be changed to a 5024-bp linear DNAfragment and slDNA should be divided into two fragments, namely, 127-bpof slDNA and double-stranded linear DNA whose length was unknown (FIG.15). After addition of 2 μg of ColE1-AfaI digest, which was composed of29, 46, 60, 62, 69, 99, 120, 126, 130, 243, 252, 323, 413, 415, 564,778, 950, 976 and 991-bp fragments, the samples were separated on 12%polyacrylamide gel electrophoresis. The lower half of the gel was cutand stained by ethidium bromide, and the gel of the region between120-bp and 130-bp was recovered and sliced. The DNAs were extracted fromthe gel by incubation at 60° C. for 30 min with the addition ofsterilized water. After centrifugation, insoluble impurities wereremoved from the supernatant by glasswool column. The supernatant wasthen lyophilized. Finally, the recovered DNAs were suspended in 20 μl ofTE buffer.

11-4 Detected of the slDNA by PCR Method

The detection of the slDNA in the samples prepared in example 11-3 wascarried out by PCR method. Primer 1871 and primer 20261 shown as SEQ IDNos. 16 and 17 in the sequence listing, respectively, were prepared todetect the slDNA. Primer 1870 shown as SEQ ID No. 18 in the sequencelisting was prepared to detect the containing plasmid DNA (FIG. 16). Theefficiencies of amplification with the two combinations of primers,1871/20261 or 1871/1870, were checked by examining the lowestconcentrations of the plasmid DNA which can be detected by PCR. As aresult, the lowest values of the concentrations detectable wereapproximately 2.5×10⁻²⁰ moles of template DNA in the 50 μl reactionmixtures in both cases.

PCR reactions were carried out with 50 μl reaction volumes in which1/100 volumes of PstI-digested DNA obtained in the example 11-3 wasincluded as the template DNA. 25 cycles of the following steps wereperformed: at 94° C. for 1 min, at 55° C. for 1 min and at 72° C. for 1min. After the reactions, 5 μl of the products were analyzed by 6%polyacrylamide gel electrophoresis, stained with ethidium bromide anddetected by FMBIO-100 (Takara Shuzo). In both cases, DNAs recovered fromthe cells transformed by pYES2-8-106 or pYES2-9-106 were amplified withthe combination of the primers 1871 and 20261. However, no fragmentswere amplified with primers 1871 and 1870. The latter observationsshowed that the amplified fragments detected in the former were notderived from the contaminating plasmid DNA but from the slDNAsynthesized in the yeast cells.

The slDNAs can be expressed from other eukaryotes identifiedhereinafter.

From these results, it was clearly demonstrated that slDNA can besynthesized in eucaryotic cells like yeasts. Furthermore, the fact thatthe transformant containing pYHS2-9-106 produced slDNA suggested thatslDNA was synthesized by means of not only leading strand synthesis butalso lagging strand synthesis.

As will be apparent to one skilled in the art in light of the teachingof the foregoing disclosure, many modifications, alterations and/orsubstitutions are readily possible in the practice of the inventionwithout departing from the spirit or scope thereof. Means which aresubstantially equivalent in terms of methodology and genetic constructswhich achieve the same objective to over-produce slDNAs independentlyfrom plasmid replication--or other competent vehicles--are intended tobe within the spirit and scope of the invention.

Example 12 Production of slDNA Independently of Plasmid Replication

12-1 Construction of Plasmid pHS2870

The 106-NdeI sequence which consists of sequences shown as SEQ. ID Nos.6 and 7 in the sequence listing was inserted at the NdeI site of pUC19.The resulting plasmid was named pUC106A. Plasmid pUC106Ad.P.O. wasconstructed by deletion of the PvuII fragment containing the lacpromoter-operator region from pUC106A. Next, Fragment RNA1870 wasprepared by PCR with RNAIIA primer shown as SEQ ID No. 19 in thesequence listing 1870 primer shown as SEQ ID No. 20 in the sequencelisting, and pUC106Ad.P.O. as a template. RNAII primer is annealed at5'-end of replication primer RNAII region and the 1870 primer isannealed at 3'-end of 106-NdeI sequence. Since each primer has a XbaIsite at 5'-end, the resulting RNA1870 fragment has XbaI sites at bothends. Next, RNA1870 fragment was digested with XbaI, and inserted intoXbaI site under lac promoter-operator region of pSTV28 (Takara ShuzoCo., Ltd. Otsu, Shiga, 520-21, Japan). The resulting plasmid was namedpSH2870. FIG. 17 shows a protocol for construction of pHS2870.

FIG. 18 shows the resulting pHS2870. As shown in FIG. 18 on plasmidpSH2870, the primer RNA sequence is placed under control of the lacpromoter and inverted repeat sequence is placed under p15A-Ori, which isa replication origin of the plasmid itself.

12-2 Determination of Amount of slDNA from Cells Harboring pHS2870

E. coli JM109 was transformed by pHS2870, and the transformant was namedJM109/pHS2870. This transformant was cultured in L-broth containing 100μg/ml chloramphenicol one vessel with 2 mM of IPTG and one without. DNAwas prepared by alkaline-SDS method from each culture and treated withRNaseA. These DNA samples were applied on the 6% polyacrylamide gelelectrophoresis and the gel was stained with ethidium bromide. As aresult, it was determined that the yield of slDNA from the cells whichwere cultured with IPTG was 100 times greater than from cells culturedwithout IPTG.

The slDNAs were determined to have a loop of about 4 bases, an overhangof 15-18 bases and a stem of about 440 bases.

As described above, the synthesis of slDNA was controlled independentlyof plasmid replication by controlling production of primer RNA.

When a plasmid is used which uses primase for primer RNA synthesis, theexpression of the primase is controlled, thus producing slDNAindependently of plasmid replication. For initiation by primases, asimilar function is performed by the gene 4 protein of phage T7, and thegene 41 and 61 proteins of phage T4. See DNA Replication, Kronberg,cited herein, Chapter 11-10.

Example 13 Inhibition of Gene Expression by Triple Helix Forming slDNAin E. coli

13-1 Construction of pHS2870GT

The duplexed oligonucleotide described in Example 12 which is composedof oligonucleotides shows as SEQ ID Nos. 8 and 9 in the sequence listingwas inserted into HindIII site of pHS2870 (FIG. 19). The direction ofthe HindIII fragment was confirmed by DNA sequencing and a plasmid whichproduces GT37 sequence containing slDNA was named pHS2860GT (FIG. 19).

13-2 Induction of slDNA Expression by IPTG(isopropylthio-β-D-galactoside

E. coli JM109 was transformed with either pHS2870 or pHS2870GT andincubated in 2 ml of L-broth (containing 50 μg/ml chloramphenicol) at37° C. overnight. This culture was diluted 10² times with L-broth(containing 50 μg/ml chloramphenicol) and IPTG (final concentration 1mM) was added when the OD₆₀₀ of the culture reached ˜0.7 and thenincubated another 2 hr. at 37° C. slDNA was isolated from the culture byalkaline-SDS method and analyzed by polyacrylamide gel electrophoresis(FIG. 20). The expression of slDNA (˜480 bp: pHS2870, ˜500 bp:pHS2970GT) induced by IPTG was approximately twenty-fold greater thanwithout IPTG.

The slDNAs were determined to have a loop of 53 bases, a stem of about440 and an overhang of 15-18 bases.

13-3 Production of E. coli MC4100λpFTR1F

E. coli MC4100λpF13, which is λpF13, see Gene, 57, p. 89-99 (1987).λpF13 which is lysogenic MC4100, was transformed with pMSTR37 (ATCC No.35695). λpF13 (is available from the Institute for Virus Research KyotoUniversity, Sakyo-ku, Kyoto 606 Japan. By in vivo recombination, λpFTR1which has a target sequence for triple helix forming slDNA in thepromoter region of β-galactosidase gene was isolated. MC4100λpFTR1F' wasproduced by lysogenization of MC4100 with λpFTR1 followed byintroduction of F' plasmid by conjugation with E. coli Nova Blue(Novegen) (FIG. 21).

13-4 Detection of the Expression Level of β-galactosidase byPulse-labelling and Immunoprecipatation

E. coliMC4100λpFTR1F' was transformed with either pHS2870 or pHS2870GTand incubated in 2 ml of L-broth (containing 50 μg/ml chloramphenicoland 15 μg/ml tetracycline) at 37° C. overnight. These overnight cultureswere diluted 50 times with modified M9 medium (1×M9 salts, 0.2%glycerol, 1 MM MgSO₄, 0.1 mM CaCl₂, 0.001% thiamine-HCl, 40 μg/mlarginine, 5 μg/ml common amino acids except methionine and cysteine!)and incubated at 37° C. IPTG (final concentration 1 mM) was added whenthe OD of the culture reached ˜0.2 and incubation was continued another3 hrs. at 37° C. 0.5 ml of each culture was labeled with 407 kBq of ³⁵S-Met, Cys (43.5 TBq/mmol, Express ³⁵ S! Protein Labeling Mix, NEN) for30 sec. at 37° C. and reaction was stopped by addition of 0.5 ml of 20%TCA. The precipitate recovered by centrifugation was washed with 0.5 mlof acetone and then dissolved in 50 mM Tris-HCl pH 8.0, 1% SDS, 2 mMEDTA. Radioactivity of the labeled protein was measured by liquidscintillation counter and 1×10₆ cpm of each sample was incubated with 1μl of anti-β-galactosidase antibody (COSMO BIO, AB-986) in 0.5 ml ofTBS-0.1% Triton X-100 at 4° C. overnight. To recover theantigen-antibody complex, 20 μl of IgG Sorb (The Enzyme Center; IGSL10)was added to the reaction mixture and incubated for 1 hr. at 4° C. withstrong agitation. The final precipitate was recovered by centrifugationand washed three times with TBS-0.1% Triton X-100, then once with 10 mMTris-HCl pH 8.0. The final precipitate was resuspended in 20 μl of SDSgel-loading buffer and heated for 1 min. at 95° C. Immunoprecipitatedsamples were separated by SDS-polyacrylamide gel electrophoresis andanalyzed by BIO IMAGE ANALYZER BAS2000 (FUGI PHOTO FILM) (FIG. 22). Thesignal intensity of the β-galactosidase band in IPTG treatedMC4100λpFTR1F' transformed with pHS2870GT was about 50% weaker than thattransformed with pHS2870.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

The inventors have made the fundamental discovery that part of thegenome is directly duplicated from the genome. The mechanism discoveredrequires neither an RNA intermediate nor reverse transcriptase (RT) asis well known. See Weiner et al., Ann. Rev. Biochem., 55. 631 (1986);Kornberg, in DNA Replication (W. H. Freeman and Company, San Francisco,Calif., 1980), pp. 101-166. New and useful DNA structures namedstem-loop DNAs (slDNAs) have been discovered.

By way of introduction, the invention provides several embodiments. Oneembodiment is a method (or process) for synthesizing a novel and usefulssDNA molecule (or structure). Another embodiment is an in vivo and invitro system for synthesizing such molecule which includes a DNAfragment which contains a suitable priming site, an inverted repeat (IR)and other necessary components for synthesizing the slDNAs.

An in vivo system for synthesizing such molecules uses a competentself-replicating vehicle and other components of the system. Anotheraspect of this embodiment is the self-replicating vehicle which containsan inverted repeat, a DNA to serve as template for the replication of afirst strand of the slDNA, a suitable priming site for the template tostart DNA synthesis in the opposite orientation and when needed, thesecond strand of the parent DNA and other components further described.

Another embodiment is the novel ss-slDNA molecules.

The invention provides a method for synthesizing a novel single-strandedDNA (ssDNA) molecule. The molecule comprises a stem-loop structure(slDNA) which stem is constituted of duplexed DNA of annealedcomplementary bases within the ssDNA, which stem forms, at one end, the5' and 3' termini of the ssDNA molecule and at the other end, asingle-stranded loop of DNA joining the opposite ends of the duplexedDNA. The method is carried out in a system which contains theconventional components of DNA synthesis, and the following components:

(a) a template DNA containing a suitable priming site and an invertedrepeat (IR) downstream of said priming site,

(b) a primer for the template to allow the start of DNA polymerization,and

(c) a DNA polymerase to replicate the sldna from the template.

The method comprises the steps of:

(1) priming the DNA template to allow the start of DNA polymerization,

(2) synthesizing the ssDNA from the primer using one of thedouble-strand of the DNA as template thereby forming one strand,continuing the DNA synthesis of the strand into the IR sequence andallowing the synthesis to cease within the IR sequence,

(3) allowing complementary bases within the newly synthesized strand atthe IR sequence to anneal forming, at one end, a loop of a non-duplexedregion and a duplexed region, the duplexed region functioning as apriming site for continued DNA synthesis,

(4) resuming DNA synthesis using as template the newly synthesizedstrand and/or the other strand of the DNA,

(5) forming the slDNA, and

(6) separating and isolation the slDNA.

The method is carried out in a system which contains all the necessaryconventional components for DNA synthesis. These components may bepresent inherently as when the method is carried out in vivo; they willnormally be introduced into the system when the method is carried out invitro.

The method calls for the presence of a DNA which has a suitable primingsite and an inverted repeat downstream of the priming site. The DNAserves as a template for directing the DNA replication. The primer canbe any oligonucleotide (whether occurring naturally or producedsynthetically) which is capable of acting to initiate synthesis of aprimer extension product which is complementary to a nucleic acidstrand. The method of initiation of DNA claims is of course well known.See Watson, Molecular Biology of the Gene, 3rd Ed., W. A. Benjamin,Inc.; DNA Synthesis: Present and Future, Molineux and Kohiyama, Eds.,(1977) Part V, "G4 and ST-1 DNA Synthesis In Vitro" by Wickner; PartVII, "DNA Synthesis in Permeable Cell Systems from Saccharomycescerevisiae" by Oertel and Goulian. Different polymerases may contributeto the synthesis of the second strand, as opposed to that contributingto the synthesis of the first strand. The primer may be a DNA or RNAprimer. Synthesis is induced in the presence of nucleotides and an agentof polymerization, such as DNA polymerase at a suitable temperature andpH.

The method of the invention uses an agent for polymerization such as anenzyme, which catalyzes the synthesis of the strand along the template.Suitable enzymes for this purpose include, for example, any DNApolymerase like E. coli DNA polymerase I, or III, Klenow fragment of E.coil DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, reversetranscriptase (RT), vital polymerases, including heat-stable enzymes.Any polymerase that will recognize its site of initiation for DNAreplication is appropriate. Generally, when the process is carried outin vivo, these genetic elements will be present; if not or if performedin vitro, they will be added to the system.

Any polymerase that will recognize the site of initiation may cause orcontribute to the polymerization. While the inventors do not wish to bebound to any particular theory at this time, it is not to be excludedthat the polymerase that contributed to the synthesis of the first DNAstrand also contributes to synthesize the other or second DNA strand.The method thus provides continuing the synthesis of the second stranduntil it terminates at terminus 3' beyond the 5' terminus of the firstformed strand. Thus, there is formed the duplexed stem of the slDNA.

Information on DNA polymerases is available. For instance, see Kornberg,in DNA Replication (W. H. Freeman and Company, San Francisco, Calif.,1980), pp. 101 166, Chapter 4, DNA Polymerase I of E. coli, Chapter 5,Other Procaryotic Polymerases, Chapter 6, Eucaryotic DNA Polymerases andChapter 7.

Other polymerases, such as those that are useful in second-strandsynthesis in cDNA may be considered.

In a specific illustration described above, it is postulated that thepolymerases are two: DNA polymerase III and polymerase I.

When it is desired to replicate a desired or target nucleic acidsequence which is capable of encoding a protein, e.g., a gene as part ofthe synthesized slDNAs, the sequence will be positioned upstream of theIR. For example, when the replication takes place in a replicationvehicle like a vector, e.g., pUCK106, which has an origin of replication(OR), the target nucleic acid sequence will be positioned in between theIR and the OR.

The method of the invention uses a double-stranded DNA fragment whichhas, as described, a priming site and also an inverted repeat (IR),i.e., two copies of an identical sequence present in the reverseorientation. The IR may be present as a sequence or "palindrome".

As will be described hereinafter, certain enzymes will be preferred overothers, such as RT when it is desired to favor introducing random pointmutations in a nucleic acid sequence.

The synthesis of the first strand proceeds in a contiguous manner alongthe dsDNA template through the IR resulting in the replication of theentire plasmid genome. At a certain frequency, the synthesis of the DNAstrand is ceased within the IR, forming a short region of sequence whichis complementary to itself, forms a loop and at the IR sequence annealsto itself. This double-stranded region within the newly synthesizedstrand is recognized as the priming site for the second or other DNAstrand. Synthesis of the second strand starts using the first formedstrand and/or the other parent strand of the DNA as template. Thus,template switching is believed to occur.

As the second strand is synthesized by the polymerase incorporating thenucleotides, the stem is formed of the annealed complementary basesresulting in its duplexed structure with internal complementarity.

The synthesis of the second strand proceeds all the way past the firstnucleotide of the first synthesized strand through the RNA primer ofthis first strand which eventually degrades, thus providing a 3'overhang. In one specific illustration, the DNA synthesis is believed toterminate at the terH site by a mechanism similar as described inDasgupta et al., Cell, 51, 1113 (1987).

By appropriate manipulations the length of the 3' end overhang can becontrolled, e.g., lengthened, as by moving the terH site downstream fromthe priming site.

Further, instead of a stem with a 3' end overhang, it is consideredfeasible to block the synthesis of the second strand before the end ofthe first strand by placing an appropriate termination site, e.g., terHupstream of the priming site. Thus, it is contemplated that eitherstrand can be longer by a predetermined length with respect to theother.

As the synthesis of the second strand ceases, the template and theformed slDNA separate.

The method of the invention may be repeated in cycles as often as isdesired. If any of the necessary components are becoming depleted, theyare resupplied as it becomes necessary.

When the method is carried out in vivo there is provided a suitablecompetent replicating vehicle which carries the necessary template DNAfragment having a priming site and an IR downstream of the priming site.The DNA fragment carrying the IR will normally be inserted into arestriction site unique in a polylinker sequence. A plasmid has beenused in which the polylinker has an IR (and symmetrical restrictionsites). When not inherently present, the DNA fragment will be providedwith a primer to the DNA sequence; the polymerase may be indigenous tothe vehicle or not. The vehicle will contain all other necessaryelements for replication and forming the slDNAs.

From the foregoing it will be understood that any self-replicatingvector which contains a DNA fragment to serve as template, an IRsequence, the necessary elements to prime and continue the replicationof the strand that will form the slDNA, is suitable to synthesize theslDNAs.

Another major embodiment of the invention provides the new ss-slDNAs.These structures have already been described hereinabove. Additionaldescription is provided hereinafter.

The new structure which has been named "stem-loop" or "slDNA" issingle-stranded. An illustration of an slDNA is shown in FIG. 5E (frompUCK106) and FIG. 7 (from pUC7).

Typical slDNAs contain a duplexed double-stranded stem of annealedcomplementary bases and a loop of a single-strand of nucleotidesconnecting the two strands of the stem. The single-strandedness of theloop is another interesting feature of the slDNAs of the invention.slDNAs can include loop of a very short sequence of nucleotides orconsiderably longer ones. The slDNAs may contain a nucleotide sequencejust long enough to form the loop and base pairing forming a shortduplexed double-strand. The minimum size should allow for base pairingenough to provide for priming site for the start of synthesis of thesecond strand. The minimum size of the loop may be limited by thestrains on the bases that would prevent their pairing into a stablestructure. The maximum size is influenced by the use intended for theslDNAs. The loop illustrated in FIG. 5B is constituted of four bases;loops of 10, 20 or more bases can be conceived. The single-strandednessof the loop of the slDNAs is a feature which may be quite useful in theutilities proposed for the slDNAs.

Yet another interesting structure contemplated for the slDNAs is thedouble slDNAs. In this structure, the free ends of the single-strands oftwo slDNAs are ligated. The structure is expected to be extremelystable. On exposure to conditions which normally denature DNAs, thesessDNAs are likely to "snap-back" to their original structure. Joining ofthe strands will be carried out by conventional procedures with DNAand/or RNA ligases. Such structures can also carry selected genes forencoding proteins and their thus provide interesting new practicalpossibilities.

It is believed that the stability, an important property of the slDNAs,tends to increase with longer duplexed tails; thus, such structures arefavored when this is a property which is to be emphasized. It should benoted that all slDNAs generated from a single replicating vehicle arenot necessarily identical in size. In the illustration shown above,slDNA from pUCK106, the 5' end of the first strand appears to beheterogeneous, some strands starting from the +1 position (whichcorresponds to the origin of ColE1 replication) (see Tomizawa et al.,Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), while other strands startfrom -1, +2 and +3. Thus, the DNAs may be considered as family ofanalogous slDNAs.

It may be noted that the presence of one or more mismatch in the DNAfragment beyond the IRs does not adversely affect the synthesis nor thestructure of the slDNAs. This is illustrated by the mismatch T for G, inthis case 25 nucleotides away from the center of the palindrome (seeFIG. 2). This mismatch was repaired in the synthesis of the slDNA.

Several utilities for the slDNAs of the invention are proposed hereinwhich take advantage of the ssDNA overhang of one end of the tail overthe other. It will therefore be appreciated that this is an importantfeature of the new structures of the invention.

The synthesis of the slDNAs in the illustrated plasmid is descriptive ofa best mode of the invention. However, any vector which contains the IRand other components described herein is suitable for synthesizing theslDNAs.

IR is a structure frequently by occurring in procaryotes and ineucaryotes. Any such IR may be used in the invention. IR sequences mayalso be prepared synthetically. An illustration is the IR synthesizedand shown in FIG. 2

Sequences of IR or palindrome sequences have been obtained from E. coli(Gilson et al., Nucl. Acids. Res., 18, 3941 (1990)); Gilson et al.,Nucl. Acids Res., 19 1375 (1991) reported palindromic sequences from E.coli and Salmonella enteritica (a palindromic unit sequence 40nucleotides long). Chalker et al., Gene, 71, (1):201-5 (1988) reportsthe propagation of a 571-bp palindrome in E. coli; inverted repeats arereported by Lewis et al., J. Mol. Biol. (England), 215, (1):73-84 (1990)in Bacillus subtilis (a 26-base pair repeat), and Saurin, Comput. Appl.Biosci., 3, (2):121-7 (1987) discusses the use of a new computer programto search systematically for repetitive palindromic structures in E.coli. The following U.S. Pat. Nos. disclose palindromic sequences:4,975,376; 4,863,858; 4,840,901; 4,746,609; 4,719,179; 4,693,980 and4,693,979.

The slDNAs can be constructed to have loops and stems of varying sizeswith tails (or overhangs at the 3' or 5' end) that fit best the purposeintended for the slDNAs. The stem may be constructed to be 100,preferably at least 300 bases to over 5,000, preferably to about 4,000bases long. The overhang of one terminus over the other can vary from afew nucleotides, e.g. 15-18, 15 to 30 or longer as of about 50 to 100 ormore bases. Since the single-stranded overhang may contain a DNAantigene fragment, it will be generally advisable that the tail overhangof the 3' or 5' terminus be rationally sized in relationship to the sizeof the DNA antigene fragment so that it may function optionally, be itas an antisense or for forming a triple helix. Similar considerationshould best be taken into account when sizing the loop of the slDNA.Loops varying in sizes from 4 to 80 can be considered. For practical andother considerations, it appears that loops of 40 to about 60 aresatisfactory.

Palindromes have been defined to include inverted repetitious sequencesin which almost the same (not necessarily the same) sequences run inopposite direction. Though some are short (3-10 bases in one direction),others are much longer, comprising hundreds of base pairs. Watson,Molecular Biology of the Gene, 3rd Ed., pps. 224-225.

The IR in the DNA fragment can vary considerably in size. Withoutintending to be limited slDNAs with inverted repeats of 10 to 30 orlonger nucleotides may be considered. Inverted repeats have beenreported to contain more than 300-bp. Current Protocols, Section 1.4.10.

The slDNAs can be the synthesis product of procaryotic or eucaryotichost expression (e.g., bacterial, yeast and mammalian cells).

Examples of appropriate vectors such as a plasmid and a host celltransformed thereby are well known to one skilled in the art. Amongappropriate hosts for plasmid carrying the necessary components areprocaryotes and eucaryotes. Procaryotes include such microorganisms asthose of the genus Escherichia, in particular E. coli; of the genusBacillus, in particular, B. subtilis. Eucaryotes include such organismsas yeast, animal, and plant, and also include such microorganisms asanimal and plant cells.

Plasmids capable of transforming E. coli include for example, the pUCand the ColE1 type plasmids. See U.S. Pat. No. 4,910,141. Plasmidscapable of transforming E. coli include for example, ColE1 type plasmidsin general. Other appropriate plasmids for transforming E. coli include:pSC101, pSF2124, pMB8, pMB9, pACYC184, pACYC177, pCK1, R6K, pBR312,pBR313, pML2, pML21, ColE1AP, RSF1010, pVH51, and pVH153.

Plasmids capable of transforming B. subtilis include: pC194, pC221,pC223, pUB112, pT127, pE194, pUB110, pSA0501, pSA2100, pTP4, pTP5 andtheir derivatives. Plasmids capable of transforming both B. subtilis andE. coli are described in J. Bacteriol., 145, 422-428 (1982); Proc. Natl.Acad. Sci. USA, 75, 1433-1436 (1978) and Principles of Gene Manipulation2nd Ed., Carr et al. Eds., University of Ca. Press, Berkeley, 1981, p.48.

Of special interest for carrying out the synthesis of the slDNAs ineucaryotes are plasmids capable of transforming S. cerevisiae: pMP78,YEp13, pBTI1, pLC544, YEp2, YRp17, pRB8 (YIp30), pBTI7, pBTI9, pBTI10,pAC1, pSLe1, pJDB219, pDB248 and YRp7. Also to be considered are YIp5,pUC-URA3, pUC-LEU2 and pUC-HIS3. See page 285 and pages 373-378 inMethods in Enzymology, Vol. 194, "Guide to Yeast Genetics and MolecularBiology", edited by Guthrie and Fink (1991), Academic Press, Inc. Otheryeast vectors are described at pages 100-104 in ExperimentalManipulation of Gene Expression, edited by Masayori Inouye, AcademicPress, Inc. (1983).

Further, of particular interest are shuttle vectors which can be used totransform E. coli as well as yeast like S. cerevisiae. Such vectorsinclude the following: pKB42 and pYC1. Other examples are listed in thesection on "Cosmid Vectors for Low and Higher Eucaryotes" in A PracticalGuide to Molecular Cloning, 2nd Edition by Bernard Perbal (1988), Wileyand Sons. Other suitable vectors are described in Vol. 2, Sections13.4.1, 13.4.2 (1989), Current Protocols. Other suitable vehiclesinclude such popular multicopy vectors like YEp24 (Botstein et al.,Gene, 8, 17 (1979)) and pJDB207 (Beggs, Genetic Engineering (Ed.Williamson), Vol. 2, p. 175, Academic Press (1982)). Others that may beselected include plasmids of the classes YEp, like YEp51, YEp52 andpYES2.

Examples of commercially available eucaryotic vectors for carrying outthe present invention are pSVL and pKSV-10 in for example, COS, CHO andHeLa cells. Other examples are listed in A Practical Guide to MolecularCloning.

These replicable genetic vehicles contain the appropriate geneticelements for replication of the vehicle, including their origin ofreplication, promoters, etc., so that upon insertion of the invertedrepeat and other elements described herein, the slDNAs will beexpressed. However, in the improvement of the invention describedherein, namely, the over-expression of the slDNAs, it is preferred thatthe synthesis and production of slDNAs be independently controlled fromthe replication of the vehicle, e.g. vector from an independentlyinducible promoter and genetic elements different from those orresponsive to or operative with different elements than those thatgovern the replication of the vehicle.

Culturing and fermentation of the transformed hosts is carried out bystandard and conventional methods known in the art. See for example,Methods in Enzymology, Vol. 185, Gene Expression Technology (Goeddel,editor) 1990 (in particular, Growth of Cell Lines); for yeasts, seeMethods in Enzymology, Vol. 194, Guide to Yeast Genetics & MolecularBiology; growth conditions for E. coli are described in CurrentProtocols in Molecular Biology, Vol. 1, at pages 1.1.1, 1.1.2, 1.1.3,1.1.4 and 1.3.1 and Molecular Cloning: A Laboratory Manual, 2nd Editionat page 1.21 and purification of plasmid DNA is described at page 1.23,culturing growth conditions suitable for mammalian cells are describedin Current Protocols, Vols. 1 and 2 at pages 9.0.4-9.0.6, 9.1.1, 9.1.3,9.2.5, 9.4.3, 11.5.2, 11.6.2 and 11.7.3.

In summary, when the necessary components for initiating and thesynthesis of the single first strand are supplied in a replicatingvehicle, namely, a DNA template fragment with an initiation site, and anIR (and the polymerase(s)), a slDNA is expected to be formed.

When the synthesis of the slDNAs of the invention is performed in vitro,the synthesis will be performed in a medium which includes conventionalcomponents for the synthesis of nucleotide strands using the DNAfragment as template. Generally, the synthesis will take place in abuffered aqueous solution, preferably at a pH of 7-9. Preferably, amolar excess of the oligonucleotides over the DNA template strand. Thedeoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also acomponent of the synthesis mixture. Heating and then cooling of thesolution is performed. The polymerase is then added and the synthesisproceeds. These components are called "conventional components" for thepurpose of the invention described herein.

Oligonucleotide synthesis may be carried out by a number of methodsincluding those disclosed in U.S. Pat. No. 4,415,734, and in Matteuci etal., J. Am. Chem. Soc., 103 (11):3185-3191 (1981), Adams et al., J. Am.Chem. Soc., 105 (3):661-663 (1983), and Bemcage et al., TetrahedronLetters, 22 (20):1859-1867 (1981).

The methods of the present invention make use of techniques or geneticengineering and molecular cloning. General techniques of geneticengineering and molecular cloning are included in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, 1990, A Practical Guide to Molecular Cloning, 2nd Ed.,Bernard Perbal (1988), Methods in Enzymology, Volume 68, Recombinant DNA(Wu, editor), Academic Press, N.Y., 1979, Methods in Enzymology, Volume185, Gene Expression Technology (Goeddel, editor) 1990, CurrentProtocols in Molecular Biology, Vols. 1, 2 and 3.

The slDNAs of the invention have several interesting utilities. Themethod of the invention may be used to provide random mutations in aselected gene. Such a system is illustrated in FIG. 8 which shows thegene for β-galactosidase in a transformed vector.

The method comprises synthesizing an slDNA containing a gene of interestlocated in the stem, isolating the slDNA, cutting out the gene from theslDNA, cloning it into an appropriate replicating vehicle and expressingthe protein encoded by the gene. The proteins can be tested for thedesired activity. By standard procedures the colonies can be screened toidentify those with mutated genes.

An illustration for generating and identifying mutations proceeds asfollows. Into pUCK19 (See Example 1) which harbors the lacZ gene, thereis ligated the 215-bp DNA fragment consisting of DNAs shown as SEQ IDNos. 1 and 2 in the sequence listing respectively, containing the 35-bpIR (described in Example 1) between the DNA fragment and the OR (asshown in Example 1). The plasmid is transformed into competent E. ColiCL83 which is grown under standard conditions. Thereafter, the lacZ geneis isolated and inserted into pUCK19 (without the IR-containingfragment). pUCK19 is digested with KasI and EcoRI and the isolating lacZgene is transformed into E. coli CL83. The frequency of mutation isscored by the known in vivo test using β-gal, a colorless substrate,which is hydrolyzed to give a dark blue product. When the colonies arecolorless, this indicates that no β-galactosidase has been produced.

Other genes of lac gene family, e.g., lacY or lacA, or other suitablegenes can be used for this screening test. The gene encoding the protein(or polypeptide) of interest may also be used to determine the frequencyof mutations and selection of appropriate gene encoding the derivedprotein (or polypeptide).

This screening procedure permits selection of competent vectors otherpolymerases that are more likely to introduce or increase the rate ofmutation in the target gene. Thus, a selected gene such as the lacZ genecan be used as the screening gene. Reverse transcriptase known to haveless replication fidelity, appears to be an enzyme of choice for thepurpose introducing mutations in a target gene encoding a desiredprotein.

The desired target protein(s) is then expressed by a competent selectedtransformed host carrying the mutated gene and selected for its desiredbiological properties.

The frequency of mutation is believed to be influenceable by selectingappropriate enzymes that are known to have less fidelity in replication.Thus, when target DNA fragments or genes are amplified, the systemdepends on the degree of replication fidelity or the infidelity that is,the frequency of error made by the DNA polymerases in replicating theinserted DNA fragment or gene. Thus for each replication error, randommutations are introduced into the genes. The higher the degree ofinfidelity of the DNA polymerase, the greater the number of mutatedgenes, and vice versa.

In one above-illustrated embodiment of the invention in which it isbelieved the PolIII and PolI were active, it is expected that the formerhas less fidelity, since PolI is known to have a self-correctingfunction. Thus, the replicating fidelity of the system can be regulatedby appropriate selection of the DNA polymerases. Generally, it isbelieved that random mutations are more likely to be introduced by thepolymerase synthesizing the second strand. An interesting candidatewould be RT.

Genes carrying the desired mutation(s) are useful in chromosomalcross-over. By this method the genes of interest or the slDNA carryingthe mutated gene of interest can be made to integrate and exchangegenetic information from one similar molecule to another. The mutatedgene locates within the genome a sequence that is similar to the vectorsequence and the homologous gene is replicated by the mutated gene.

In this manner there can be generated new strains of microorganisms (orhosts) that contain the mutated gene and will express a desired protein.The slDNA carrying the mutated gene can be made in vitro or in vivo.

The polymerase chain reaction (PCR) is a rapid procedure for in vitroenzymatic amplification of a specific segment of DNA. The standard PCRmethod requires a segment of double-stranded DNA to be amplified, andalways two-single stranded oligonucleotide primers flanking the segment,a DNA polymerase, appropriate deoxyribonucleoside triphosphate (dNTPs),a buffer, and salts. See Current Protocols, Section 15.

The ss-slDNA of the invention can be amplified with a single primer.This feature considerably simplifies the amplification, helps toovercome the problem of one primer finding its proper initiation site,renders it more economical and helps overcome problems associated withthe traditional PCR method.

The amplification of the slDNA comprises denaturing the slDNA to form asingle-stranded DNA (from 3' to 5' ends). The reaction follows the usualsequence: priming from 3' end, polymerase reaction, denaturing andannealing. It is carried out for 25 cycles leading to million-foldamplification of slDNA. The slDNA can be carrying a gene for encoding atarget protein.

A recent report in Science, 252, 1643-1650 (Jun. 21, 1991) entitled"Recent Advances in the Polymerase Chain Reaction" by Erlich et al.discusses problems associated with primers and improvements that arebeing proposed to the PCR method.

Accordingly, a method for amplification of the ss-slDNA structures ofthe invention by a method using one primer is of great interest. TheslDNA could be carrying a gene of interest, such as a mutated genehaving improved biological properties.

The in vivo production of the slDNAs of the invention can be somanipulated to provide a desired sequence. The slDNAs so produced maythen be used as antisense DNA.

A fascinating utility that is being considered is the role that slDNAsof the invention can play on the formation of triple helix DNA, ortriplex DNA, and the resulting new triplex slDNA structures. A recentreport in Science, 252, 1374-1375 (Jun. 27, 1991), "Triplex DNA FinallyComes of Age", highlights the timeliness of the present invention.Triplex DNA can be formed by binding a third strand to specificrecognized sites on chromosomal DNA. Synthetic strands of sizespreferably containing the full complement of bases (such as 11-15 andhigher), are discussed. The slDNAs of the invention with long 3' (or 5'ends (and the loop of non-duplexed bases) would appear to be excellentcandidates. The resulting triplex DNA is expected to have increasedstability and usefulness. New therapies based on the triple helixformation, including in AIDS therapy and selective gene inhibition andothers are proposed in the Report.

In an important embodiment of the invention, the slDNA comprise in theirsingle-stranded portion, an antigene which is a DNA sequence whichregulates gene function as an antisense fragment or a sequence whichforms a triple helix.

slDNA containing such DNA sequence regulating or controlling genefunction as an antisense sequence and a sequence with the ability oftriple helix formation (hereinafter referred to "antigene") can beproduced and used in vivo as a DNA molecule regulating gene function byway of the present invention.

The most effective manner to regulate the expression of a specific geneis to act on the gene directly.

The present invention provides methods for the regulation of geneexpression, for example, by utilizing antisense RNA, antisense DNA, orthe DNA with the ability of triple helix formation. As for antisense RNAor DNA, they anneal with the target mRNA because they are complementaryto the target gene, and they inhibit the translation of protein frommRNA by double-stranded formation. Antisense RNA can be produced in acell by a promoter, but it is difficult to produce antisense DNA in vivobecause of its single-stranded DNA. So by way of the present invention,the synthetic DNA, as antisense DNA, is provided into the cell toregulate gene expression.

As for the DNA with the triple helix formation, the present inventionprovides for the third single-stranded DNA to bind to double-strandedDNA which has polypurine (G or A) at one strand and polypyrimidine (C orT) at the other strand by Hoogsteen binding activity. This thirdsingle-stranded DNA has the ability of triple helix formation.. Its manyapplications are suited not only for gene regulation but also the fieldof gene technology. Like antisense DNA, in vivo production ofsingle-stranded DNA with the ability of triple helix formation is verydifficult and therefore, by way of the present invention, the syntheticDNA is provided into the cell to regulate gene expression. In vitroproduction of antisense DNA or DNA with the ability of triple helixformation is provided for by way of a DNA synthesizer. However, it isnot known of the artificial production of single-stranded DNA in vivoexcept for msDNA. But msDNA is a single-stranded cDNA transcribed frommRNA by reverse transcriptase in a cell, and therefore its practicalapplication is very difficult because of its complex synthetic process.On the other hand, the slDNA of the present invention containing thesequence for gene regulation can be produced in vivo without an mRNAintermediate and can be useful for gene regulation.

When there is an inverted repeat sequence (IR sequence), the firststrand DNA synthesized anneals to another template DNA at the IRsequence, and then the second strand DNA synthesis begins. The DNAsynthesis proceeds to the origin of DNA duplication and is terminated atthe terminate site of transcription. Finally, this single-stranded DNAseparates as slDNA from the original template DNA. The loop region and3' end or 5' end of slDNA form a single strand. So it is possible by wayof the present invention for the antisense DNA or DNA with the abilityof triple helix formation to be inserted into these single-strandedregions. slDNA with an antisense DNA sequence anneals with the targetRNA to inhibit gene function, and furthermore, the target RNA is cleavedby cellular RNase H activity. As slDNA with the ability of triple helixformation is also produced at gene duplication, it is always produced invivo. This enables the regulation of the target double-stranded DNA. Themethod of the invention for insertion of antisense DNA or DNA with theability of triple helix formation into slDNA is described in detail,hereinafter.

To insert antigene DNA into slDNA, in the embodiment of using a plasmid,the IR sequence should be on the way to the direction of plasmidduplication and the antigene sequence should be cloned between two IRsequences. After transformation, the cell containing this plasmidproduces antigene slDNA continuously as the plasmid duplicates. slDNAhas overhang structure at the 5' end or 3' end because, in the processof slDNA synthesis, the 5' end priming site of slDNA is different fromthe 3' end terminal site. Therefore, it is possible to insert antigeneinto these overhang regions of slDNA. To produce the slDNA whichcontains antigene at the 3' or 5' end, the antigene sequence should becloned between the priming site and the terminate site of slDNA from theplasmid with the IR sequence. And after transformation, single-strandedDNA with antigene at the 5' or 3' end of slDNA is produced.

Originally, slDNA was discovered in E. coli. The slDNA can be alsoexpressed in yeast. The slDNA of the present invention is also usefulfor antigene production and gene regulation in a eucaryote, such as amammal.

It can be seen that the present invention provides a significantcontribution to the arts and science.

As described hereinabove, the slDNAs are valuable genetic constructs.There is therefore an important need to produce such slDNAs in yieldsheretofore not yet attained.

The present invention provides a process and the genetic constructs bywhich slDNAs are expressed in high yields. In the process of the priordescribed embodiments, slDNA levels of production were dictated andlimited by the endogenous level of primer that initiated replicationfrom the origin of replication of the recombinant DNA vehicle. Inaccordance with this embodiment of the invention, a system has beenconstructed wherein for best results, the recombinant DNA constructcomprises a group of elements which operate independently of the geneticelements that control the replication of the vehicle. In this system--orgroup of elements--the cellular level of primers for slDNAs synthesishas been increased dramatically, thereby initiating synthesis andexpression of the valuable slDNAs in high yields.

slDNAs can be expressed in prokaryotes or in eukaryotes. The method ofexpressing the cells comprises culturing a prokaryotic or eukaryotichost transformed with a recombinant DNA self-replicatable vehiclecapable of directing expression of slDNA. The recombinant DNA vehiclecomprises the following elements operatively linked: A DNA sequencecoding for an inducible promoter, preferably a strong promoter-operatorwhich upon induction produces an RNA primer or a protein, e.g. an enzymelike primase, which functions to initiate synthesis of slDNA utilizingthe appropriate substrates and DNA polymerase endogenous to the hostcell. The vehicle comprises further an inverted repeat downstream of theDNA sequence encoding the RNA primer or primase. Further, therecombinant DNA vehicle comprises an origin of replication whichfunctions in a replication of the DNA vehicle itself and a selectablemarker, e.g. a gene conferring resistance to an antibiotic.

In the preferred embodiment of the invention, initiation of slDNAsynthesis is not coupled to or dependent upon the replication of therecombinant DNA vehicle. However, it is also contemplated that arecombinant DNA vehicle can be constructed such that the primer whichinitiates slDNA synthesis also initiates replication of the plasmid.

When it is desired to over-express slDNAs which include in theirsingle-stranded portion an antigene fragment, the replicable vehiclecomprises the antigene fragment either between the two opposite invertrepeat sequences or between the priming site utilized to initiate slDNAsynthesis and the inverted repeat. slDNAs carrying the antigene fragmentare thus produced in high yields.

Useful inducible promoters to drive expression of the DNA fragment whichcodes for the RNA primer or primase protein include, for instance, theearly or late promoters of SV40, the lac promoter, the trp promoter, thetac or TRC promoters, the major promoters of phage λ e.g. λpl or λpr,etc.; the promoter of fd coat protein, the promoter fore-phosphoglycerate kinase or other glycolytic enzymes, the promoter ofacid phosphatase, the promoters of the yeast mating factors, and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses. When it is desired to express theslDNAs in mammalian cells, it is additionally possible to amplify theexpression units by linking the gene to that coding for dehydrofolatereductase and applying a selection to those cells which results inamplification of the desired gene.

Useful promoters are inducible by an appropriate inducing agent. Forexample, the lac and tac promoters are induced by IPTG; malidixic acidis commonly used to induce the expression of the λpl or λpr promoters.Other such combinations are known in the art. See for instance, DNAReplication, Kornberg, cited below. Current Protocols in MolecularBiology, Ausubet et at. John Wiley & Sons, Inc. 1994 (CurrentProtocols).

When it is desired to over-express slDNAs in eukaryote cells like yeastcells, as shown above or mammalian cells, the scheme described abovewill be used separating the system to replicate the yeast cells fromthat synthesis the slDNA, thus causing the slDNAs expressed from theyeast cells to be expressed at a higher level and rate independentlyfrom the replication of the yeast cells. For a variety of suitabletechniques and yeast molecular biology, see, Guide to Yeast Genetics andMolecular Biology, Ed. Guthrie and FInk, Academic Press, Inc., Methodsin Enzymology, Volume 194, 1991 and other references cited herein. Alsosee Experimental Manipulation of Gene Expression, Ed. Masayori Inouye,Academic Press, 1983 in general and Chapter 5, Vectors for High Level,Inducible Expression of Cloned Genes in Yeast.

FIGS. 17 and 18 illustrate a protocol to construct a preferred constructof the invention. As shown in FIG. 17, the priming site which functionsto initiate slDNA synthesis is derived from an origin of replication ofa plasmid e.g. pUC19 in FIG. 17. However, in its final form, thispriming site does not function in the replication of the recombinant DNAvehicle encoding slDNA, instead the recombinant DNA vehicle contains itsown functional origin of replication.

Other origins of replication can also be used to direct slDNA synthesisincluding for instance pMB1, ColE1, pSC101, R6-5, mini F, R1, R100, R6K,Rts1, RK2, P1, ColEII, ColEIII, p15A, T4 bacteriophage, T7bacteriophage, RSF1030, pBR, and pUC.

Some of these origins of replication may, as is known in the art,require proteins for efficient activity. If the priming site whichinitiates synthesis is derived from such an origin, it is within thescope of the invention that the inducible promoter may in thesesituations induce and direct expression of that protein. It iscontemplated that the priming site can be derived from other origins ofreplication, different from that shown in FIG. 17. For example, otherorigins of replication which can be used include pMB1, ColE1, pSC101,R6-5, mini F, R1, R100, R6K, RTs1, RK2, P1, ColEII, ColEIII, p15A,RDF1030, T4 bacteriophage, T7 bacteriophage, pBR, and pUC. In addition,it is also within the scope of the invention that the functional originof replication which directs the replication of the recombinant DNAvehicle is not limited to p15A as shown in FIG. 17, but may include forexample, pMB1, ColE1, pSC101, R6-5, mini F, R1, R100, R6K, RTs1, RK2,P1, ColEII, ColEIII, T4 Bacteriophage, T7 Bacteriophage, p15A, RSF1030,pBR, and pUC. Various other origins of replication are known in the art.DNA Replication, Oxford University Press 1991; DNA Replication,Kornberg, W. H. Freeman and Company, 1980.

There are quite a number of suitable starting plasmids other than pUC106which have suitable origins of replication like the pMB1, ColE1, mini-F,R1, R100, R6K, Rts1, RK2, F, P1, T4 Bacteriophage, T4 Bacteriophase,ColEII, ColEIII and others.

Methods for culturing the host transformed or transfected with the DNArecombinant vehicle are well known. They comprise culturing the hostunder appropriate conditions of growth and collecting the desired slDNAfrom the culture. See, Current Protocols, cited herein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

A preferred embodiment of the invention can readily be described byreference to FIGS. 17 and 18.

As can be observed from FIG. 17, the construction of the recombinant DNAvehicle pHS2870, comprises a functional origin of replication p15A, i.e.a region of the plasmid which encodes initiation primers RNAI RNAII andthe ROP protein, which together function in the replication of theplasmid. As is seen, p15A Ori region originated from pSVT28.

This set of elements is responsible for the replication of the plasmiditself and does not direct the synthesis of the slDNAs. This function isperformed by a set of elements that directs the synthesis of the slDNAsindependently from the replication of the plasmid. This set of elementscomprises an inducible strong promoter-operator which upon inductionproduces a primer positioned upstream of the inverted repeat. Further,adjacent to the promoter-operon, and downstream thereof, the plasmidcarries a DNA fragment encoding an RNA primer e.g. RNAII or a proteine.g. primase as discussed above to start the synthesis of the slDNAs.Downstream of this DNA fragment there is positioned an inverted repeatwhich as described above, participates in the synthesis and constitutespart of the slDNA. When it is desired that the slDNA carry an antigene,that antigene is positioned upstream of the inverted repeat anddownstream the DNA encoding the primer, i.e. between these two elements,or between the two opposite segments of the inverted repeats e.g.inserted at a suitable restriction site. The antigens sequence, asdiscussed hereinabove, is a fragment which can anneal to a target mRNAand inhibit mRNA translation to protein or binds to dsDNA, therebyforming a triple helix which inhibits the expression of target DNA.

As is apparent from this description, the elements which operate in thesynthesis of slDNAs may originate from different genetic constructs thanto those which control the replication of the plasmid, but this need notbe necessarily so.

As is taught herein, there is provided a general scheme for theconstruction of a plasmid which synthesizes slDNA independently fromplasmid replication. In accordance with this scheme, there is insertedinto any desired replicatable vehicle, any DNA fragment which containsan inverted repeat of the desired length which will participate in thesynthesis of the slDNAs and constitute the stem of the slDNAs. It istherefore possible in accordance with the invention to regulate thereplication of the slDNAs independently and in excess of the synthesisof the plasmid itself.

It is important to note that the invention is not limited to theparticular constructs illustrated or the constructs from which thefunctional elements originate. Nor, is the invention limited toparticular plasmids. One can select other plasmids or vehicles that willsupply the necessary genetic elements for the final construct.

Nor is the invention limited to the particular genetic elementsillustrated. Following the general concept of the invention, one skilledin the art will readily construct the appropriate means to implementthat concept of the invention.

A number of embodiments of the invention have been describedhereinbefore. It is apparent from the description that other embodimentswhich utilize the teaching of their invention can be utilized which willreach substantially the same result of overexpressing a single-strandedmolecule like slDNA.

REFERENCES

1. Weiner et al., Ann. Rev. Biochem., 55, 631 (1986)

2. Inouye and Inouye, TIBS, 16, 18 (1991a)

3. Inouye and Inouye, Ann. Rev. Microbiol., 45. 163 (1991b).

4. Higgins et al., Nature, 298, 760 (1982)

5. Gilson et al., EMBO. J., 3, 1417 (1984)

6. Gilson et al., Nucl. Acids Res., 19, 1375 (1991)

7. Gilson et al., Nucl. Acids Res., 3941 (1990)

8. Simmett et al., J. Biol. Chem., 266, 8675 (1991)

9. Tomizawa et al., Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)

10. Molecular Cloning, A Laboratory Manual, Sambrook et al., 2d Ed.(Sections 1.121-1.40)

11. Kornberg, in DNA Replication (W. H. Freeman and Company, SanFrancisco, Calif., 1980), pp. 101-166, and Chapter 4, DNA Polymerase Iof E. coli, Chapter 5, Other Procaryotic Polymerases, Chapter 6,Eucaryotic DNA Polymerases and Chapter 7.

12. Watson, Molecular Biology of the Gene, 3rd Ed., W. A. Benjamin, Inc.

13. DNA Synthesis: Present and Future, Molineux and Kohiyama, Eds.,(1977) Part V, "G4 and ST-1 DNA Synthesis In Vitro" by Wickner; and PartVII, "DNA Synthesis in Permeable Cell Systems from Saccharomycescerevisiae" by Oertel and Goulian

14. Dasgupta et al., Cell, 51, 1113 (1987)

15. Chalker et al., Gene, 71, (1):201-5 (1988)

16. Lewis et al., J. Mol. Biol. (England), 215, (1):73-84 (1990)

17. Saurin, W., Comput. Appl. Biosci., 3, (2):121-7 (1987)

18. U.S. Pat. Nos. 4,975,376; 4,863,858; 4,840,901; 4,746,609;4,719,179; 4,693,980 and 4,693,979

19. Current Protocols, Section 1.4.10

20. U.S. Pat. No. 4,910,141

21. J. Bacteriol., 145, 422-428 (1982)

22. Proc. Natl. Acad. Sci. USA, 75, 1433-1436 (1978)

23. Principles of Gene Manipulation 2nd ed., Carr et al. Eds.,University of Ca. Press, Berkeley, 1981, p. 48

24. Methods in Enzymology, Vol. 194, "Guide to Yeast Genetics andMolecular Biology", edited by Guthrie and Fink (1991), Academic Press,Inc., pps. 285 and 373-378

25. Experimental Manipulation of Gene Expression, edited by MasayoriInouye, Academic Press, Inc. (1983), pps. 100-104

26. A Practical Guide to Molecular Cloning, "Cosmid Vectors for Low andHigher Eucaryotes", 2nd Edition by Bernard Perbal (1988), Wiley and Sons

27. Current Protocols, Vol. 2, Sections 13.4.1, 13.4.2 (1989)

28. Botstein et al., Gene, 8, 17 (1979)

29. Beggs, Genetic Engineering (ed. Williamson), Vol. 2, p. 175,Academic Press (1982)

30. Methods in Enzymology, Vol. 185, Gene Expression Technology(Goeddel, editor) 1990 (in particular, Growth of Cell Lines)

31. Current Protocols in Molecular Biology, Vol. 1, at pages 1.1.1,1.1.2, 1.1.3, 1.1.4 and 1.3.1

32. Current Protocols, Vols. 1 and 2 at pages 9.0.4-9.0.6, 9.1.1, 9.1.3,9.2.5, 9.4.3, 11.5.2, 11.6.2 and 11.7.3

33. U.S. Pat. No. 4,415,734

34. Matteuci et al., J. Am. Chem. Soc., 103 (11):3185-3191 (1981)

35. Adams et al., J. Am. Chem. Soc., 105 (3):661-663 (1983)

36. Bemcage et al., Tetrahedron Letters, 22 (20):1859-1867 (1981)

37. Methods in Enzymology, Volume 68, Recombinant DNA (Wu, R., editor),Academic Press, N.Y., 1979

38. Current Protocols in Molecular Biology, Vols. 1, 2 and 3

39. Current Protocols, Section 15

40. Science, 252, 1643-1650 (Jun. 21, 1991) entitled "Recent Advances inthe Polymerase Chain Reaction" by Erlich et al.

41. Science, 252, 1374-1375 (Jun. 27, 1991), "Triplex DNA Finally Comesof Age"

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 20                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTAGAGATATGTTCATAAACACGCATGTAGGCAGATAGATCTTTGGTTGTGAATCGCAAC60                CAGTGGCCTTATGGCAGGAGCCGCGGATCACCTACCATCCCTAATGACCTGCAGGCATGC120               AAGCTTGCATGCCTGCAGGTCATTAGGTACGGCAGGTGTGCTCGAGGCGAAGGAGTGCCT180               GCATGCGTTTCTCCTTGGCTTTTTTCCTCTGGGAT215                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTAGATCCCAGAGGAAAAAAGCCAAGGAGAAACGCATGCAGGCACTCCTTCGCCTCGAGC60                ACACCTGCCGTACCTAATGACCTGCAGGCATGCAAGCTTGCATGCCTGCAGGTCATTAGG120               GATGGTAGGTGATCCGCGGCTCCTGCCATAAGGCCACTGGTTGCGATTCACAACCAAAGA180               TCTATCTGCCTACATGCGTGTTTATGAACATATCT215                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGTTATCCACAGAATCAG18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGCTTACTAGTCATACTCTTCCTTTTTCAATGCTAGCA38                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGCTTGCTAGCATTGAAAAAGGAAGAGTATGACTAGTA38                                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TATGGATATGTTCATAAACACGCATGTAGGCAGATAGATCTTTGGTTGTGAATCGCAACC60                AGTGGCCTTATGGCAGGAGCCGCGGATCACCTACCATCCCTAATGACCTGCAGGCATGCA120               AGCTTGCATGCCTGCAGGTCATTAGGTACGGCAGGTGTGCTCGAGGCGAAGGAGTGCCTG180               CATGCGTTTCTCCTTGGCTTTTTTCCTCTGGGACA215                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TATGTCCCAGAGGAAAAAAGCCAAGGAGAAACGCATGCAGGCACTCCTTCGCCTCGAGCA60                CACCTGCCGTACCTAATGACCTGCAGGCATGCAAGCTTGCATGCCTGCAGGTCATTAGGG120               ATGGTAGGTGATCCGCGGCTCCTGCCATAAGGCCACTGGTTGCGATTCACAACCAAAGAT180               CTATCTGCCTACATGCGTGTTTATGAACATATCCA215                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCTTTTGGTGGGTGGGTGGGTGGGTGTTGTGTGGGTGGGTGGGTTTTA49                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AGCTTAAAACCCACCCACCCACACAACACCCACCCACCCACCCACCAAA49                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCGAGGCCTCCCTCCCTCCCTCCCTCTTGACACCCTCCCTCCCATTTGTTATAATGTGTG60                GA62                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AGCTTCCACACATTATAACAAATGGGAGGGAGGGTGTCAAGAGGGAGGGAGGGAGGGAGG60                CC62                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATCCTGATGCCTGCTCTGCG20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TGGCCAGAGAGAGAAAGAGAAGAAGAAAAGATCTTAGCATACGATTTAGGTGACACTATA60                GAATACACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA120               AGCTTGCGGCCGCATCCCTATAGTGAGTCGTATTACGATGGGCCCTCCCTCCTCTCCCCT180               CCTCCCTCGAGGCCT195                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TGGCCAGAGAGAGAAAGAGAAGAAGAAAAGATCTTAGCATACGATTTAGGTGACACTATA60                GAATACACAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCG120               AATTCGCGGCCGCATCCCTATAGTGAGTCGTATTACGATGGGCCCTCCCTCCTCTCCCCT180               CCTCCCTCGAGGCCT195                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGTCTAGATCCCAGAGGAAAAAAG24                                                    (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TGATCCGCGGCTCCTGCCATAAGG24                                                    (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGTCTAGAGATATGTTCATAAAC23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AGATCTAGAGCAAACAAAAAAACCACCG28                                                (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GGTCTAGATCCCAGAGGAAAAAAG24                                                    __________________________________________________________________________

We claim:
 1. A recombinant, self-replicating DNA molecule forover-expressing stem-loop DNA ("slDNA") in a compatible host whichcomprises the following elements: a strong inducible promoter-operatorand adjacent and downstream thereof, a DNA fragment encoding a primerwhich initiates the synthesis of slDNA, and downstream thereof aninverted repeat, which elements operate to replicate slDNA independentlyfrom the replication of the molecule, and an origin of replication whichdirects the replication of the DNA molecule independently from thesynthesis of the slDNA.
 2. The DNA molecule of claim 1 wherein theprimer which is produced upon induction of the promoter anneals only tothe origin of replication encoded by the DNA fragment upstream of theinverted repeat.
 3. The DNA molecule of claim 2 wherein the geneticelements which cause replication of the DNA molecule originate from adifferent genetic molecule than the genetic elements which causesynthesis of the slDNA.
 4. The DNA molecule of claim 1 wherein oneorigin of replication directs replication of the molecule and anotherorigin of replication which is encoded by the DNA fragment directssynthesis of the slDNA.
 5. The DNA molecule of claim 4 wherein theprimer which is encoded by the DNA fragment is an RNA primer.
 6. The DNAmolecule of claim 1 wherein the primer which is encoded by the DNAfragment is primase.
 7. The DNA molecule of claim 4 wherein the primerfor the origin of replication of the replication elements for the slDNAis an RNAII primer.
 8. The DNA molecule of claim 3 wherein the origin ofreplication which directs the replication of the molecule is derivedfrom a plasmid other than a pUC type plasmid.
 9. The DNA molecule ofclaim 1 wherein the vehicle is a plasmid.
 10. The DNA molecule of claim9 wherein the plasmid is an E. coli plasmid.
 11. The DNA molecule ofclaim 1 which comprises a foreign DNA antigene sequence positionedeither between the DNA fragment encoding the primer and the invertedrepeat or in the inverted repeat between the two opposite sequenceshereof.
 12. The DNA molecule of claim 11 wherein the DNA antigene ispositioned in the inverted repeat.
 13. A method for improving theexpression of an slDNA comprising culturing a host transformed with arecombinant DNA molecule which causes expression of a stem-loop DNA inhigh yields in the prokaryotic host independently from the replicationof the DNA molecule, the DNA molecule comprising the following elements:a strong inducible promoter-operator and adjacent and downstreamthereof, a DNA fragment encoding for a primer which initiates thesynthesis of slDNA, and downstream thereof an inverted repeat, whichelements operate to replicate slDNA independently from the replicationof the molecule, and an origin of replication which directs thereplication of the DNA molecule independently from the synthesis of theslDNA.
 14. The method of claim 13 wherein the primer which is producedupon induction of the promoter anneals only to the origin of replicationencoded by the DNA fragment upstream of the inverted repeat.
 15. Themethod of claim 13 wherein the genetic elements which cause replicationof the DNA molecule originate from a different genetic molecule than thegenetic elements which cause synthesis of the slDNA.
 16. The method ofclaim 13 wherein one origin of replication directs the replication ofthe molecule and another origin of replication encoded by the DNAfragment directs the synthesis of the slDNA.
 17. The method of claim 15wherein the primer which is encoded by the DNA fragment is an RNAprimer.
 18. The method of claim 13 wherein the primer which is encodedby the DNA fragment is primase.
 19. The method of claim 13 wherein theorigin of replication which directs the replication of the molecule isderived from a plasmid other than a pUC type plasmid.
 20. The method ofclaim 13 wherein the RNA primer is an RNA II primer.
 21. The method ofclaim 15 wherein the genetic elements which operate to replicate the DNAmolecule function independently from the genetic elements which functionto synthesize the slDNA.
 22. The method of claim 13 wherein the DNAmolecule comprises a foreign DNA antigene sequence positioned eitherbetween the DNA fragment encoding the primer and the inverted repeat orin the inverted repeat between the two opposite sequences thereof. 23.The method of claim 22 wherein the DNA antigene is positioned in theinverted repeat.
 24. A prokaryotic host transformed with the recombinantmolecule of any one of claims 2, 3, 4, 5, 8 and 9.